Direct activation of gastric H,K-ATPase by N-terminal protein kinase C phosphorylation. Comparison of the acute regulation mechanisms of H,K-ATPase and Na,K-ATPase

Abstract

In this study we compared the protein kinase dependent regulation of gastric H,K-ATPase and Na,K-ATPase. The protein kinase A/protein kinase C (PKA/PKC) phosphorylation profile of H,K-ATPase was very similar to the one found in the Na,K-ATPase. PKC phosphorylation was taking place in the N-terminal part of the alpha-subunit with a stoichiometry of approximately 0.6 mol Pi/mole alpha-subunit. PKA phosphorylation was in the C-terminal part and required detergent, as is also found for the Na,K-ATPase. The stoichiometry of PKA-induced phosphorylation was approximately 0.7 mol Pi/mole alpha-subunit. Controlled proteolysis of the N-terminus abolished PKC phosphorylation of native H,K-ATPase. However, after detergent treatment additional C-terminal PKC sites became exposed located at the beginning of the M5M6 hairpin and at the cytoplasmic L89 loop close to the inner face of the plasma membrane. N-terminal PKC phosphorylation of native H,K-ATPase alpha-subunit was found to stimulate the maximal enzyme activity by 40-80% at saturating ATP, depending on pH. Thus, a direct modulation of enzyme activity by PKC phosphorylation could be demonstrated that may be additional to the well-known regulation of acid secretion by recruitment of H,K-ATPase to the apical membranes of the parietal cells. Moreover, a distinct difference in the regulation of H,K-ATPase and Na,K-ATPase is the apparent absence of any small regulatory proteins associated with the H,K-ATPase.

FIGURE 1

PKA and PKC phosphorylation profiles of gastric H,K-ATPase-containing microsomes. Autoradiogram of ³²P-labeled proteins after SDS-gel electrophoresis showing PKA (lanes 1 and 2) and PKC (lanes 3 and 4) phosphorylation of pig gastric microsomes in the absence (lanes 1 and 3) or in the presence (lanes 2 and 4) of 0.1% Triton X-100. Membranes were incubated with protein kinase buffer, in the presence of 3 μCi/pmol [³²P]ATP, as mentioned in Experimental Procedures. A total of 2 μg of phosphorylated proteins was separated by SDS-PAGE and visualized by autoradiography. The H,K-ATPase α-subunit was excised from the gel and the associated radioactivity measured by scintillation counting (Cornelius and Logvinenko, 1996). In the PKA profile with TX-100 (lane 2) the H,K-ATPase α-subunit, Rap 1A, and histone H3 are identified. In the PKC profile the H,K-ATPase α-subunit, PKC, and histone H1 are phosphorylated both in the absence and presence of Triton X-100, although the phosphorylation intensity of the H,K-ATPase α-subunit and the autophosphorylation of PKC are enhanced by detergent (see Fig. 4).

FIGURE 2

Comparison of the effects of sub-cmc concentrations of C₁₂E₈ on catalytic activity of Na,K-ATPase and H,K-ATPase. Pig stomach membranes were treated with 20 μM of C₁₂E₈ and the K⁺-activation curves were measured for control and C₁₂E₈ treated-membranes. The hydrolytic activity was measured as described in Methods. The reaction mixture contained 30 mM histidine, pH 7.00, 1 mM MgCl₂, 0.3 μg protein, and the indicated KCl concentrations. Activities are expressed as percentage of maximum activity in controls.

FIGURE 3

N-terminal truncation of H,K-ATPase. A shows a Coomassie-stained SDS-PAGE demonstrating N-terminal truncation of the H,K-ATPase α-subunit by controlled proteolysis. Microsomes were incubated without (lane 1) or with (lanes 2 and 3) trypsin as described in Experimental Procedures. Lane 1, control; Lane 2, membranes were incubated in a proteolysis buffer, pH 7.2 containing 20 mM K⁺; Lane 3, membranes were incubated in a proteolysis buffer, pH 6.2, and in the absence of K⁺. Washed membranes were subjected to SDS-PAGE and the gel was stained with Coomassie blue. Representation of two independent experiments is shown. As seen, after mild proteolysis the mobility increases as a result of the N-terminal truncation. B shows K⁺ activation of control and N-terminal truncated enzyme in the presence of 3 mM ATP, pH 7.2. The fitted curves are hyperbolic with apparent K⁺ affinities of 0.36 ± 0.08 mM and 0.38 ± 0.09 mM, respectively. C shows ATP activation of H,K-ATPase before and after truncation at 20 mM K⁺, pH 7.2. In both cases the activation is biphasic with high-affinity apparent K_0.5 ∼40 μM, and low-affinity components which saturated in the mM range.

FIGURE 4

PKA and PKC phosphorylation profiles of native and N-terminal truncated H,K-ATPase in the absence or presence of 0.1% Triton X-100. The upper panel shows autoradiogram of PKA phosphorylated H,K-ATPase preparations and the lower panel PKC phosphorylated H,K-ATPase preparations. Phosphorylated proteins were separated by SDS-PAGE and analyzed by autoradiography. The radioactivity associated with the α-subunits was measured by scintillation counting and the phosphorylation stoichiometry calculated as previously described (Cornelius and Logvinenko, 1996). For PKA phosphorylation the following phosphorylation stoichiometries were found (mol Pi/mol α): control-TX-100, 0.09 ± 0.014; control+TX-100, 0.71 ± 0.04; Truncated-TX-100, 0.018 ± 0.005; Truncated+TX-100, 0.30 ± 0.034. For PKC phosphorylation the stoichiometries were control-TX-100, 0.63 ± 0.01; control+TX-100, 1.16 ± 0.012; Truncated-TX-100, 0.13 ± 0.014; Truncated+TX-100, 0.43 ± 0.017.

FIGURE 5

Alignment of amino acid sequences of H,K-ATPase α-subunit N-terminus (upper) and C-terminus (lower) from different species. Putative phosphorylation sites are labeled with the respective amino acid number in the full-length mature protein. Sequences were obtained form GenBank. In the N-terminus a PKC site at Ser-27 is conserved. In the C-terminus, PKC sites at the M5M6 hairpin (Ser-785) and at the M8M9 loop (Thr-949) are conserved and in close apposition to the likewise conserved PKA site at Ser-953.

FIGURE 6

Proteolytic fingerprinting to localize C-terminal PKC phosphorylation. (A) An immunoblot using a specific H,K-ATPase α-subunit antibody of intact (left lane) and extensively trypsin treated (right lane) H,K-ATPase. Nearly all H,K-ATPase α-subunit is cleaved and several low molecular weight proteolytic products are probed with the antibody. (B) An autoradiogram showing proteolytic fingerprinting of PKC phosphorylated H,K-ATPase in the absence (left lane) or in the presence (right lane) of 0.1% TX-100. In the absence of TX-100 PKC phosphorylation is mainly associated with the 32-kDa N-terminal fragment, whereas in the presence of TX-100 additional phosphorylated fragments appear, most notably one at 19 kDa. A faint band migrating at an apparent molecular mass of 12 kDa is also observed. (C) Autoradiogram comparing PKA phosphorylation and PKC phosphorylation of H,K-ATPase in the absence (lanes 1 and 3) and presence (lanes 2 and 4) of 0.1% TX-100 after proteolytic fingerprinting. As seen, PKA and PKC phosphorylation are detected at a 19-kDa fragment and only in the presence of TX-100.

FIGURE 7

(A) Time course of PKC phosphorylation of H, K-ATPase. The figure shows the phosphorylation stoichiometry of PKC phosphorylation of H, K-ATPase as a function of time. PKC phosphorylation was performed as described in Methods except that the reaction was terminated after the indicated time intervals with SDS sample buffer containing 0.5% TCA. Volumes containing 2 μg protein were loaded onto the gel. After electrophoresis and autoradiography (shown as inset to the figure), the α bands were cut from the gel and counted for radioactivity. The phosphorylation stoichiometry was calculated as described in Methods. (B) Autoradiogram showing absence of endogenous phosphatase activity in H,K-ATPase preparation. A total of 4 μg of H, K-ATPase membranes were phosphorylated by PKC for 30 min at 24°C in the presence of ³²P[ATP] as described in Methods. The reactions were then quenched with 2.5 mM cold ATP and the phosphorylated membranes mixed with increasing concentrations of unphosphorylated membranes (0, 1, 2, 3, and 4 μg as indicated in the figure) and incubated again for 30 min. The samples were then treated with SDS sample buffer and subjected to SDS-PAGE and autoradiography. The constant level of phosphorylation indicates the absence of phosphatase activity.

FIGURE 8

Steady-state K⁺ activation of hydrolytic activity at low ATP. H,K-ATPase membranes were incubated in PKC buffer at 24°C, pH 7.5, in the presence (□) or absence of PKC (○) for 30 min followed by measurement of the steady-state hydrolytic activity at variable K⁺ concentrations in the presence of 10 μM ATP in histidine buffer with the pH values adjusted to 6.5 or 7.2 as indicated. At both pH = 6.5 (A) and pH = 7.2 (B), identical K⁺ activation curves were obtained in control and after PKC phosphorylation. Note, however, the higher apparent K⁺-affinities (K_0.5 ∼0.23 mM at pH 6.5 vs. 0.08 mM at pH 7.2) and the lower maximum hydrolytic activities (∼14 μmol·mg⁻¹·h⁻¹ at pH 6.5 vs. ∼7 μmol·mg⁻¹·h⁻¹ at pH 7.2) when pH increases.

FIGURE 9

Steady-state K⁺ activation of hydrolytic activity at high ATP. These experiments were performed by incubation of H,K-ATPase membranes in PKC buffer at 24°C in the presence (□) or absence of PKC (○) for 30 min followed by measurement of the maximum hydrolytic activity at variable K⁺ concentration at 3 mM ATP. Incubation of the enzyme with PKC at both pH 6.5 (A) and 7.2 (B) resulted in a significant activation of the hydrolytic activity. Curves represent fitting of the data with a hyperbolic binding curve. At pH 7.2 the maximum hydrolytic activity at 30°C was 63.5 ± 1.4 μmol·mg⁻¹·h⁻¹ in control experiments whereas in membranes pre-incubated with PKC the activity was 112.5 ± 1.2 μmol·mg⁻¹·h⁻¹ (P < 0.007). A similar activation was also observed at pH 6.2: 57.5 ± 1.9 μmol·mg⁻¹·h⁻¹ for control and 87.7 ± 1.4 μmol·mg⁻¹·h⁻¹ after PKC phosphorylation (P < 0.003). The fitted values for the K_0.5 of K⁺-activation at pH 7.2 were 0.59 mM and 0.58 mM in control and after PKC phosphorylation, respectively. At pH 6.2 the K_0.5 of K⁺-activation were identical 1.7 mM before and after PKC phosphorylation.

SCHEME 1

FIGURE 10

The steady-state ATP activation curve obtained at [K⁺] = 10 mM, pH 7.2 in control H,K-ATPase membranes (○) and after PKC phosphorylation (□). In control the activation is biphasic, whereas after PKC phosphorylation it becomes hyperbolic and the maximum hydrolytic activity increased significantly.